Clostridium botulinum produces a characteristic botulinum neurotoxin (BoNT), which is classified by the Centers for Disease Control and Prevention as one of the six highest-risk threat agents for bioterrorism (“Category A” bioterrorism agents). BoNT is a 150 kDa single chain protein which is linked by a disulfide bond which can be cleaved into a 100 kDa Heavy Chain (HC) and a 50 kDa Light chain (LC) by endogenous or exogenous proteases. The LC is a zinc metalloprotease which cleaves different SNARE proteins depending on the serotype causing flaccid paralysis. The HC consists of a C-terminal binding domain (HC) which is responsible for receptor binding and a N-terminal translocation domain (HN) which is responsible for delivering the catalytic light chain to the neuronal cytosol. Based on their ability to be neutralized by type specific antiserum, BoNTs have traditionally been categorized into seven serotypes (BoNT/A-G), among which BoNTs A, B, E and F are known to cause human botulism. Clostridium botulinum type A (BoNT/A) is of particular importance and interest due to its implication in human botulism cases, and its potential for use as a bioterrorism agent. It is also the serotype most commonly used in the medical field to treat muscle movement disorders and in the cosmetic industry to smooth facial wrinkles bringing large profits to the pharmaceutical industry.
There are currently five different type A subtypes identified as A1, A2, A3, A4 and A5 (Carter et al., BMC Genomics 10:115 (2009); Dover et al., J. Clin. Microbiol. 47(7):2349-2350 (2009); Jacobson et al., Appl. Environ. Microbiol. 77(12):4217-4222 (2011)). BoNT/A1 is the most studied and commonly used for the clinical purposes. BoNT/A1 and BoNT/A5 are very similar: both containing a HA neurotoxin gene cluster, while BoNT/A2, BoNT/A3 and BoNT/A4 contain an OrfX neurotoxin gene cluster (Jacobson et al., Appl. Environ. Microbiol. 74:2778-2786 (2008); Jacobson et al., Appl. Environ. Microbiol. 77(12):4217-4222 (2011)). Both bont/A3 and bont/A4 are located on a plasmid instead of a chromosome (Marshall et al., Biochem. Biophys. Res. Commun. 361:49-54 (2007); Smith et al., PLoS ONE 2(12):e1271 (2007)). Interestingly, BoNT/A3 could not be effectively neutralized by anti-BoNT/A1 antibody, which is the only serum currently available to treat botulism (Mazuet et al., PLoS One 5(8):e12416 (2010)).
While BoNT/A3 has interesting properties, purification of BoNT/A3 is challenging due to its low levels of production. It would be advantageous, therefore, to develop improved methods for obtaining highly pure preparations of BoNT/A3.